In the last post, I mentioned that high background on your chemiluminescent Western blot could be due to excess, pooled substrate or letting the blot dry out. So how do you keep the blot moist?
Well, you can place a clear, flat plastic covering on the blot to prevent it from drying out. This will keep the substrate in contact with the HRP enzyme and keep the blot moist. However, there are a few things to keep in mind when using a plastic covering or wrap.
- Image the plastic covering by itself first to determine if it scatters light (which would cause high background). You may need to try several types of plastic coverings before finding the best one.
- Handle the wrap and the blot with care. Wrapping the blot in plastic wrap may cause unwanted background, especially if it’s folded or handled roughly. When using plastic wrap it is important to avoid wrinkles, as they scatter light (and, again, give you high background).
- Try to avoid leaving fingerprints from pressing on the blot (we’ll talk about fingerprints in another post).
In the image, you can that there is unwanted background caused by the plastic wrap.
An alternative to plastic wrap is to use a clear, flat plastic covering, such as a clear sheet protector (from your local office supply store).
IMPORTANT: Be sure to image the sheet or any plastic you use to see if it fluoresces all by itself! After all, this is a creative use of sheet protectors, but not what the manufacturer had in mind so not what they were QC’d for.
And, remember, go digital with the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System!
You do chemiluminescent Western blots – you do TONS of them. Sometimes, as I am sure you have discovered, you have ‘challenges’ – the darn thing just doesn’t look right!
What are some things that may happen when using chemiluminescence detection for Western blotting and how do you resolve them? The next series of blog posts here will offer up some tips on how to optimize chemiluminescent Westerns. These tips were designed for Westerns detected with a digital imager, like the Odyssey Fc Chemiluminescent and Near-infrared Fluorescent Imaging System. But many will also apply if you are (still) in the darkroom and using film. 🙂
Pools of excess substrate on the membrane can lead to areas of high background, as can adding more substrate to a membrane that has dried. Apply the substrate carefully and wick off any pools of substrate before imaging. Do not allow the membrane to dry.
The image above is an example of high background due to pooled excess substrate. If your blot looks like the image below, then the membrane dried out and then someone tried to add more substrate.
Optimizing Chemiluminescent Blots on the Odyssey Fc Imager